Kentaro Ohkuni, Wei-Chun Au, Loran Gliford, Evelyn Suva, and Munira A. Basrai
- Submitter: Kentaro Ohkuni (Postdoc or fellow)
- email: kentaro.ohkuni@nih.gov
Centromeric localization of histone H3 variant, CENP-A (Cse4 in S. cerevisiae, CID in flies), is essential for faithful chromosome segregation. Overexpressed CENP-A leads to its mis-localization to non-centromeric chromatin and contributes to aneuploidy in yeast, flies and humans. Overexpression and mislocalization of CENP-A is observed in many cancers and is proposed to promote tumorigenesis. In budding yeast, ubiquitin-mediated proteolysis of Cse4 by E3 ubiquitin ligases such as Psh1, Slx5, and SCF (Skp, Cullinn, F-box) -Met30/Cdc4, prevents its mislocalization to non-centromeric chromatin. Overexpression of Cse4 (GAL-CSE4) leads to synthetic dosage lethality (SDL) in psh1∆, slx5∆, met30-6, and cdc4-1 strains. A genome-wide screen to identify mutants that exhibit SDL with GAL-CSE4 led to the identification of evolutionarily conserved Cdc48 (p97/VCP in metazoans), a chaperone of the AAA-ATPases family. Cdc48 plays an essential role in many cellular processes such as endoplasmic reticulum-associated degradation (ERAD), spindle disassembly, membrane fusion, autophagy, and transcriptional control. Here we show that Cdc48 regulates removal of CENP-ACse4 from non-centromeric regions. Growth assays showed that mutants in CDC48 and its co factors UFD1 and NPL4 exhibit SDL with GAL-CSE4. We observed an enrichment of Cse4 in chromatin and mislocalization of Cse4 to non-centromeric chromatin in cdc48-3, ufd1-2, and npl4-1 strains. Higher levels of ubiquitinated Cse4 were present in cdc48-3, ufd1-2, and npl4-1 strains. We determined that Cse4 interacts with Npl4, which has a major binding site for ubiquitin, under normal physiological conditions. We propose that Cdc48/Ufd1/Npl4 segregase regulates dissociation/eviction of mislocalized Cse4 from non-centromeric region and targets it for ubiquitin-mediated proteolysis. Our ongoing studies are aimed at defining the molecular mechanism by which Cdc48/Ufd1/Npl4 prevents stable association of mislocalized Cse4 to non-centromeric regions in yeast and human cells.